PROJECT SUMMARY/ABSTRACT Exciting progress in the field of cancer immunotherapy has generated tremendous interest in identifying new immunotherapeutic targets, with particular interest in those focused on activating macrophages against cancer. During cancer progression, malignant cells evade elimination by macrophages through the expression of ?don't eat me? signals, which inhibit phagocytosis by engaging inhibitory receptors on macrophages. One such anti- phagocytic mechanism involves the expression of CD47, which effectively shields cancer cells from macrophage-mediated clearance through interaction with the inhibitory macrophage receptor, SIRP?. Therapeutic strategies blocking CD47-SIRP? signaling have demonstrated great promise in pre-clinical studies for several cancers. Recent exciting progress has led to the discovery of a second ?don't eat me? signaling axis that involves MHC class I (MHC I) expressed by cancer cells, and the inhibitory macrophage receptor, LILRB1. Antagonism of MHC I-LILRB1 signaling has shown potential as an effective strategy to promote tumor clearance by augmenting phagocytosis of cancer cells. However, further preclinical studies are warranted to evaluate the efficacy of LILRB1 blockade as an anti-cancer therapy. The initial goal of this proposal is to enhance the knowledge of LILRB1 as an innate immune checkpoint molecule that can be targeted in cancer by 1) profiling LILRB1 expression within tumors and assessing the response to LILRB1 blockade in phagocytosis assays with primary tumor-associated macrophages (Aim 1) and 2) testing combination clinical strategies to amplify both macrophage and T-cell mediated tumor clearance (Aim 2). Lastly, this proposal aims to identify additional ?don't eat me? signals employed by human cancers that inhibit macrophage-mediated clearance. By leveraging the knowledge of established innate immune checkpoints, a list of 90 candidate genes with high likelihood to modulate the macrophage-mediated anti-tumor immune response has been compiled. The proposed work involves the implementation of a novel CRISPR/Cas9 genetic screening pipeline against this small pool of genes to identify additional ?don't eat me? signals which can be blocked therapeutically to augment macrophage-mediated cancer clearance (Aim 3). The accomplishment of the proposed aims will provide valuable insight into how to develop clinical approaches that maximize both the innate and adaptive immune responses to cancer.